A7r5 Vascular Smooth Muscle Research Articles

Fluoride impairs vascular smooth muscle A7R5 cell lines via disrupting amino acids metabolism

Given the insidious and high-fatality nature of cardiovascular diseases (CVDs), the emergence of fluoride as a newly identified risk factor demands serious consideration alongside traditional risk factors. While vascular smooth muscle cells (VSMCs) play a pivotal role in the progression of CVDs, the toxicological impact of fluoride on VSMCs remains largely uncharted. In this study, we constructed fluorosis model in SD rats and A7R5 aortic smooth muscle cell lines to confirm fluoride impaired VSMCs. Fluoride aggravated the pathological damage of rat aorta in vivo. Then A7R5 were exposed to fluoride with concentration ranging from 0 to 1200 μmol/L over a 24-h period, revealing a dose-dependent inhibition of cell proliferation and migration. The further metabolomic analysis showed alterations in metabolite profiles induced by fluoride exposure, notably decreasing organic acids and lipid molecules level. Additionally, gene network analysis underscored the frequency of fluoride's interference with amino acids metabolism, potentially impacting the tricarboxylic acid (TCA) cycle. Our results also highlighted the ATP-binding cassette (ABC) transporters pathway as a central element in VSMC impairment. Moreover, we observed a dose-dependent increase in osteopontin (OPN) and α-smooth muscle actin (α-SMA) mRNA level and a dose-dependent decrease in ABC subfamily C member 1 (ABCC1) and bestrophin 1 (BEST1) mRNA level. These findings advance our understanding of fluoride as a CVD risk factor and its influence on VSMCs and metabolic pathways, warranting further investigation into this emerging risk factor.

Mechanism of cytotoxicity induced by the cigarette smoke extract (CSE) of heated tobacco products in vascular smooth muscle cells: A comparative study of the cytotoxic effects of CSE and the ferroptosis inducer, erastin

Heated tobacco products (HTPs) are marketed worldwide as less harmful alternatives to combustible cigarettes; however, their cytotoxic mechanisms in vascular smooth muscle cells are poorly understood. Ferroptosis is defined as iron-dependent cell death caused by the accumulation of lipid peroxidation products. In this study, the cytotoxic effects of nicotine- and tar-free cigarette smoke extracts (CSE) derived from three types of HTPs and the ferroptosis inducer, erastin, on vascular smooth muscle A7r5 cells were compared. Cigarette smoke from all HTPs was generated according to the following puffing regime: 55 mL, puff volume; 30 s, puff interval; 2 s, puff duration; bell-shaped, puff profile; and no blocking of the ventilation holes. Erastin and CSE decreased mitochondrial metabolic activity and increased lactate dehydrogenase leakage. The cytotoxic effects of erastin were almost completely inhibited by the radical-trapping antioxidant, UAMC-3203; iron chelator, deferoxamine mesylate (DFO); 12/15-lipoxygenase (12/15-LOX) inhibitor, baicalein; and selective 15-LOX inhibitor, ML351. In contrast, CSE-induced cell damage was partially attenuated by UAMC-3203, baicalein, and ML351 but not by DFO. These results suggest that erastin induces ferroptosis via 15-LOX-mediated iron-dependent lipid peroxidation, whereas CSE causes iron-independent cell damage via 15-LOX-mediated lipid peroxidation-dependent and -independent mechanisms.

Ligustilide from Radix Angelica Sinensis prevents the migration of vascular smooth muscle A7r5 cells based on network pharmacology and experimental verification

ObjectiveLigustilide (Lig), a major component of Radix Angelica Sinensis, exhibits a protective effect on many cardiovascular diseases. Our previous study showed that Lig inhibited the proliferation of vascular smooth muscle cells. In this study, we aimed to investigate the effect of Lig on the migration of vascular smooth muscle A7r5 cells induced by Angiotensin II (Ang II) and explore the mechanism underlying based on network pharmacology. MethodsIn this study, scratch-wound healing assay and transwell migration assay were used to assess the migration activity of Lig on Ang II-induced vascular smooth muscle A7r5 cells, and network pharmacology method was used to predict the possible target molecules. Western blot analysis, Gelatin zymography assay, and small interfering RNA (siRNA) were used to determine the underlying mechanism of Lig on the migration in Ang II-induced A7r5 cells. ResultsWe found that Lig could prevented the migration induced by Ang II in A7r5 cells, which could be associated with c-Myc and MMPs from the results of network pharmacology. Our results revealed that Lig could significantly reduce the increase in the protein expression levels of c-Myc and MMP-2 in Ang II-induced A7r5 cells, but not affect the protein expression levels of MMP-9. Our data further showed that c-Myc siRNA, just as Lig, could obviously inhibit the cell migration accompanied by decreased MMP-2 expression. ConclusionThese findings suggested the anti-migratory effect of Lig could be associated with the c-Myc/MMP-2 pathway, and Lig administration had a promising potential for preventing cardiovascular diseases.

Chicken Muscle-Derived ACE2 Upregulating Peptide VVHPKESF Inhibits Angiotensin II-Stimulated Inflammation in Vascular Smooth Muscle Cells via the ACE2/Ang (1–7)/MasR Axis

This study aimed to evaluate the modulatory effects of four chicken muscle-derived peptides [VRP, LKY, VRY, and VVHPKESF (V-F)] on angiotensin II (Ang II)-induced inflammation in rat vascular smooth muscle A7r5 cells. Only V-F could significantly attenuate Ang II-stimulated inflammation via the inhibition of NF-κB and p38 MAPK signaling, being dependent on the Mas receptor (MasR) not on the Ang II type 1 or type 2 receptor (AT1R or AT2R). V-F accelerated Ang II degradation by enhancing cellular ACE2 activity, which was due to ACE2 upregulation other than a direct ACE2 activation. These findings demonstrated that V-F ameliorated Ang II-induced inflammation in A7r5 cells via the ACE2/Ang (1-7)/MasR axis. Three peptide metabolites of V-F─VHPKESF, PKESF, and SF─were identified but were not considered major contributors to V-F's bioactivity. The regulation of peptide V-F on vascular inflammation supported its functional food or nutraceutical application in the prevention and treatment of hypertension and cardiovascular diseases.

Sdox, a H2S releasing anthracycline, with a safer profile than doxorubicin toward vasculature

Sdox is a synthetic H2S-releasing doxorubicin (Dox) less cardiotoxic and more effective than Dox in pre-clinical, Dox-resistant tumour models. The well-known anthracycline vascular toxicity, however, might limit Sdox clinical use. This study aimed at evaluating Sdox vascular toxicity in vitro, using Dox as reference compound.Both vascular smooth muscle A7r5 and endothelial EA.hy926 cells were more sensitive to Dox than Sdox, although both drugs equally increased intracellular free radical levels. Sdox released H2S in both cell lines. The H2S scavenger hydroxocobalamin partially reverted Sdox-induced cytotoxicity in A7r5, but not in EA.hy926 cells, suggesting a role for H2S in smooth muscle cell death. Markers of Sdox-induced apoptosis were significantly lower than, in A7r5 cells, and comparable to those of Dox in EA.hy926 cells. In A7r5 cells, Dox increased the activity of caspase 3, 8, and 9, Sdox affecting only that of caspase 3. Moreover, both drugs induced comparable DNA damage in A7r5 cells, while Sdox was less toxic than Dox in Ea.hy926 cells.In fresh aorta rings, only Dox weakly increased phenylephrine-induced contraction when endothelium was present. In rings cultured with both drugs for 7 days, Sdox blunted phenylephrine- and high K+-induced contractions though at a concentration 10-fold higher than that of Dox.In conclusion, Sdox may represent the prototype of an innovative anthracycline, effective against Dox-resistant tumours, displaying a more favourable vascular toxicity profile compared to the parent compound.

Antioxidant peptides derived from hydrolysates of red tilapia (Oreochromis sp.) scale

Processing red tilapia (Oreochromis sp.) yields a large quantity of scale, a byproduct of the aquaculture industry. This study focuses on evaluating the potential of red tilapia scales as a source of antioxidant peptides. Scales were ground and enzymatically hydrolyzed by Alcalase® 2.4 L. The hydrolysate obtained was fractionated to purify and identify the antioxidant peptides by membrane ultrafiltration and chromatographic technology guided by the antioxidant activity in rat vascular smooth muscle A7r5 cells. After ultrafiltration, the fraction with molecular weight of 3–10 kDa showed the highest activity and their chemical nature was hydrophilic. The sequence of twenty antioxidant peptides, containing 6–16 amino acids, was identified by liquid chromatography–tandem mass spectrometry. The results indicate that the red tilapia (Oreochromis sp.) scale could be a new natural source of antioxidant peptides.

Spent Hen Muscle Protein-Derived RAS Regulating Peptides Show Antioxidant Activity in Vascular Cells.

Spent hens are egg-laying hens reaching the end of their egg-laying cycles, being a major byproduct of the egg industry. Recent studies have been focusing on finding new value-added uses for spent hens. We have previously identified four bioactive peptides from spent hen muscle proteins, including three angiotensin-converting enzyme (ACE) inhibitory (ACEi) peptides (VRP, LKY, and VRY), and one ACE2 upregulating (ACE2u) peptide (VVHPKESF (V-F)). In the current study, we further assessed their antioxidant and cytoprotective activities in two vascular cell lines—vascular smooth muscle A7r5 cells (VSMCs) and endothelial EA.hy926 cells (ECs)—upon stimulation by tumor necrosis factor alpha (TNFα) and angiotensin (Ang) II, respectively. The results from our study revealed that all four peptides attenuated oxidative stress in both cells. None of the investigated peptides altered the expression of TNFα receptors in ECs; however, VRY and V-F downregulated Ang II type 1 receptor (AT1R), while V-F upregulated the Mas receptor (MasR) in VSMCs. Further, we found that the antioxidant effects of VRP, LKY, and VRY were likely through acting as direct radical scavengers, while that of V-F was at least partially ascribed to increased endogenous antioxidant enzymes (GPx4 and SOD2) in both cells. Besides, as an ACE2u peptide, V-F exerted antioxidant effect in a MasR-dependent manner, indicating a possible involvement of the upregulated ACE2-MasR axis underlying its antioxidant action. The antioxidant activities of VRP, LKY, VRY, and V-F in vascular cells indicated their multifunctional properties, in addition to their ACEi or ACE2u activity, which supports their potential use as functional food ingredients against hypertension.

In vitro vascular toxicity assessment of NitDOX, a novel NO-releasing doxorubicin

The conjugation of doxorubicin (DOX) with nitric oxide (NO)-releasing groups gave rise to novel anthracyclines, such as nitrooxy-DOX (NitDOX), capable to overcome multidrug resistance. The widely described anthracycline cardiovascular toxicity, however, might limit their clinical use. This study aimed to investigate NitDOX-induced effects, as potential hazard, on vascular smooth muscle A7r5 and endothelial EA.hy926 cell viability, on the mechanical activity of freshly and cultured rat aorta rings, as well as on Cav1.2 channels of A7r5 cells. DOX was used as a reference compound.Although an increase in intracellular radicals and a reduction in mitochondrial potential occurred upon treatment with both drugs, A7r5 and EA.hy926 cells proved to be more sensitive to DOX than to NitDOX. Both compounds promoted comparable effects in A7r5 cells, whereas NitDOX was less active than DOX in inducing DNA damage and in eliciting apoptotic-mediated cell death revealed as an increase in sub-diploid-, DAPI- and annexin V-positive- EA.hy926 cell percentage. Moreover, in EA.hy926 cells, NitDOX doubled basal NO content, while preincubation with the NO-scavenger PTIO increased NitDOX-induced cytotoxicity.DOX exhibited a negligible contracturing effect in endothelium-intact rings, while NitDOX induced a significant ODQ-sensible, vasodilation in endothelium-denuded rings. In arteries cultured with both drugs for 7 days, NitDOX prevented either phenylephrine- or KCl-induced contraction at a concentration 10-fold higher than that of DOX. These results demonstrate that NitDOX displays a more favourable vascular toxicity profile than DOX. Taking into account its greater efficacy against drug-resistant cells, NitDOX is worth of further investigations in preclinical and clinical settings.

Vitamin D-regulated osteocytic sclerostin and BMP2 modulate uremic extraskeletal calcification.

We induced chronic kidney disease (CKD) with adenine in WT mice, mice with osteocyte-specific deletion of Cyp27b1, encoding the 25-hydroxyvitamin D 1(OH)ase [Oct-1(OH)ase-/-], and mice with global deletion of Cyp27b1 [global-1α(OH)ase-/-]; we then compared extraskeletal calcification. After adenine treatment, mice displayed increased blood urea nitrogen, decreased serum 1,25(OH)2D, and severe hyperparathyroidism. Skeletal expression of Cyp27b1 and of sclerostin and serum sclerostin all increased in WT mice but not in Oct-1α(OH)ase-/- mice or global-1α(OH)ase-/- mice. In contrast, skeletal expression of BMP2 and serum BMP2 rose in the Oct-1α(OH)ase-/- mice and in the global-1α(OH)ase-/- mice. Extraskeletal calcification occurred in muscle and blood vessels of mice with CKD and was highest in Oct-1α(OH)ase-/-mice. In vitro, recombinant sclerostin (100 ng/mL) significantly suppressed BMP2-induced osteoblastic transdifferentiation of vascular smooth muscle A7r5 cells and diminished BMP2-induced mineralization. Our study provides evidence that local osteocytic production of 1,25(OH)2D stimulates sclerostin and inhibits BMP2 production in murine CKD, thus mitigating osteoblastic transdifferentiation and mineralization of soft tissues. Increased osteocytic 1,25(OH)2D production, triggered by renal malfunction, may represent a "primary defensive response" to protect the organism from ectopic calcification by increasing sclerostin and suppressing BMP2 production.

Autophagy mediates tumor necrosis factor-α-induced phenotype switching in vascular smooth muscle A7r5 cell line.

Vascular smooth muscle cells (VSMC) dedifferentiation from a contractile to a synthetic phenotype contributes to atherosclerosis. Atherosclerotic tissue has a chronic inflammatory component with high levels of tumor necrosis factor-α (TNF-α). VSMC of atheromatous plaques have increased autophagy, a mechanism responsible for protein and intracellular organelle degradation. The aim of this study was to evaluate whether TNF-α induces phenotype switching of VSMCs and whether this effect depends on autophagy. Rat aortic Vascular smooth A7r5 cell line was used as a model to examine the phenotype switching and autophagy. These cells were stimulated with TNF-α 100 ng/mL. Autophagy was determined by measuring LC3-II and p62 protein levels. Autophagy was inhibited using chloroquine and siRNA Beclin1. Cell dedifferentiation was evaluated by measuring the expression of contractile proteins α-SMA and SM22, extracellular matrix protein osteopontin and type I collagen levels. Cell proliferation was measured by [3H]-thymidine incorporation and MTT assay, and migration was evaluated by wound healing and transwell assays. Expression of IL-1β, IL-6 and IL-10 was assessed by ELISA. TNF-α induced autophagy as determined by increased LC3-II (1.91±0.21, p<0.001) and decreased p62 (0.86±0.02, p<0.05) when compared to control. Additionally, TNF-α decreased α-SMA (0.74±0.12, p<0.05) and SM22 (0.54±0.01, p<0.01) protein levels. Consequently, TNF-α induced migration (1.25±0.05, p<0.05), proliferation (2.33±0.24, p<0.05), and the secretion of IL-6 (258±53, p<0.01), type I collagen (3.09±0.85, p<0.01) and osteopontin (2.32±0.46, p<0.01). Inhibition of autophagy prevented all the TNF-α-induced phenotypic changes. TNF-α induces phenotype switching in A7r5 cell line by a mechanism that required autophagy. Therefore, autophagy may be a potential therapeutic target for the treatment of atherosclerosis.

Purification and characterization of antioxidant peptides from cooked eggs using a dynamic in vitro gastrointestinal model in vascular smooth muscle A7r5 cells

Antioxidant peptides derived from food sources are considered as safer alternatives to commercially available antioxidant drugs. As one of the most abundant protein sources, hen’s egg proteins were extensively used to produce antioxidant peptides by enzymatic hydrolysis. Our previous work indicated that gastrointestinal digestion of cooked eggs significantly increased the antioxidant activity due to hydrolysis of egg proteins. To characterize the responsible antioxidant peptides, cooked eggs were digested in a simulated in vitro model of human gastro-intestinal digestion. Prepared digests were fractionated with FPLC (Fast Protein Liquid Chromatography) and RP-HPLC (Reverse-Phase High-Performance Liquid Chromatography) and the antioxidant activity was determined in A7r5 cells (vascular smooth muscle cell line). Further identification of peptides from peptide fractions with the highest antioxidant activity was carried out using LC-MS/MS. Four peptides derived from ovalbumin, DSTRTQ (48–53), DKLPG (61–65), DVYSF (96–100), and ESKPV (205–209), were identified; of which DKLPG did not show antioxidant activity in cells. Enzyme cleave analysis suggested that these four peptides were likely released from ovalbumin only by pepsin non-specific cleaves. It is postulated that egg consumption may exert protection against oxidative stress on human health due to release of antioxidant peptides during digestion.

Traumatic brain injury in vivo and in vitro contributes to cerebral vascular dysfunction through impaired gap junction communication between vascular smooth muscle cells.

Gap junctions (GJs) contribute to cerebral vasodilation, vasoconstriction, and, perhaps, to vascular compensatory mechanisms, such as autoregulation. To explore the effects of traumatic brain injury (TBI) on vascular GJ communication, we assessed GJ coupling in A7r5 vascular smooth muscle (VSM) cells subjected to rapid stretch injury (RSI) in vitro and VSM in middle cerebral arteries (MCAs) harvested from rats subjected to fluid percussion TBI in vivo. Intercellular communication was evaluated by measuring fluorescence recovery after photobleaching (FRAP). In VSM cells in vitro, FRAP increased significantly (p<0.05 vs. sham RSI) after mild RSI, but decreased significantly (p<0.05 vs. sham RSI) after moderate or severe RSI. FRAP decreased significantly (p<0.05 vs. sham RSI) 30 min and 2 h, but increased significantly (p<0.05 vs. sham RSI) 24 h after RSI. In MCAs harvested from rats 30 min after moderate TBI in vivo, FRAP was reduced significantly (p<0.05), compared to MCAs from rats after sham TBI. In VSM cells in vitro, pretreatment with the peroxynitrite (ONOO(-)) scavenger, 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron[III], prevented RSI-induced reductions in FRAP. In isolated MCAs from rats treated with the ONOO(-) scavenger, penicillamine, GJ coupling was not impaired by fluid percussion TBI. In addition, penicillamine treatment improved vasodilatory responses to reduced intravascular pressure in MCAs harvested from rats subjected to moderate fluid percussion TBI. These results indicate that TBI reduced GJ coupling in VSM cells in vitro and in vivo through mechanisms related to generation of the potent oxidant, ONOO(-).

Association of matrix Gla protein gene functional polymorphisms with loss of bone mineral density and progression of aortic calcification

Two matrix Gla protein (MGP) polymorphisms were associated with progression of aortic calcification and femoral neck bone loss in men. All these findings were also functionally corroborated in two vascular and bone in vitro systems indicating that MGP genetic variations can be partly responsible of higher risk of bone loss and vascular calcification. MGP plays an important role in bone and vascular mineralization as confirmed by MGP-deficient murine model. We therefore aimed to find a genetic association among -138T>C, -7G>A, and Thr83Ala MGP single-nucleotide polymorphisms (SNPs), bone loss, and progression of aortic calcification in a randomly selected general population of 296 individuals who participated in the European Vertebral Osteoporosis Study. To evaluate the rate of change in bone mineral density (BMD) and the progression of aortic calcification, dual X-ray absorptiometry and lateral spine X-rays were performed at baseline and after 4 years of follow-up. Genotyping for the three polymorphisms was carried out using polymerase chain reaction and restriction fragment length analysis. In addition, functional studies of MGP-7G>A and Thr83Ala SNPs were performed on transiently transfected osteoblast-like UMR-106 and vascular smooth muscle A7r5 cells. The proportion of men who had lost BMD in the femoral neck was higher among homozygous -7AA and 83Ala-Ala (p = 0.039 and p = 0.009, respectively), and also featured a higher risk of progression of aortic calcifications (OR = 5.6, 95% CI = 1.2-27.8 and OR = 6.8, 95% CI = 1.4-32.3, respectively). No effect was observed in women. The MGP-7A allele produced a reduction in luciferase activity compared to MGP-7G: 47% less in vascular cells and 34% less in bone cells (p = 0.001 and 0.012, respectively). In vascular cells under calcifying conditions, the MGP 83Thr allele showed a slightly higher, although not significant, inhibition than the MGP 83 Ala allele in calcium content suggesting functional differences between both variants. These results suggest that MGP genetic variations could predict a higher risk of bone loss and progression of vascular calcification in men.

Inositol 1,4,5-Trisphosphate (IP3) Receptor Up-regulation in Hypertension Is Associated with Sensitization of Ca2+ Release and Vascular Smooth Muscle Contractility

Resistance arteries show accentuated responsiveness to vasoconstrictor agonists in hypertension, and this abnormality relies partly on enhanced Ca(2+) signaling in vascular smooth muscle (VSM). Although inositol 1,4,5-triphosphate receptors (IP3Rs) are abundant in VSM, their role in the molecular remodeling of the Ca(2+) signaling machinery during hypertension has not been addressed. Therefore, we compared IP3R expression and function between mesenteric arteries of normotensive and hypertensive animals. Levels of IP3R transcript and protein were significantly increased in mesenteric arteries of hypertensive animals, and pharmacological inhibition of the IP3R revealed a higher contribution of IP3-dependent Ca(2+) release to vascular contraction in these arteries. Subsequently, we established cultured aortic VSM A7r5 cells as a cellular model that replicates IP3R up-regulation during hypertension by depolarizing the VSM cell membrane. IP3R up-regulation requires Ca(2+) influx through L-type Ca(2+) channels, followed by activation of the calcineurin-NFAT axis, resulting in IP3R transcription. Functionally, IP3R up-regulation in VSM is associated with enhancement and sensitization of IP3-dependent Ca(2+) release, resulting in increased VSM contraction in response to agonist stimulation.

Synthesis and studies of calcium channel blocking and antioxidant activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives

Abstract The novel 1,4-dihydropyridine derivatives containing the cationic pyridine moiety at the position 4, and the N -propargyl group as a substituent at position 1 of the 1,4-DHP cycle were designed, synthesised, and assessed in biological tests. Among all the novel compounds, the 4-( N -dodecyl) pyridinium group-containing compounds 11 (without the N -propargyl group) and 12 (with the N -propargyl group) demonstrated the highest calcium antagonistic properties against neuroblastoma SH-SY5Y (IC 50 about 5–14 μM) and the vascular smooth muscle A7r5 cell (IC 50 – 0.6–0.7 μM) lines, indicating that they predominantly target the L-type calcium channels. These compounds showed a slight total antioxidant activity. At concentrations close to those of L-type calcium channel blocking ones, compound 12 did not affect mitochondrial functioning; also, no toxicity was obtained in vivo. The N -propargyl group as a substituent at position 1 of the 1,4-DHP cycle did not essentially influence the compounds’ activity. The 4-( N -dodecyl) pyridinium moiety-containing compounds can be considered as prototype molecules for further chemical modifications and studies as cardioprotective/neuroprotective agents.

P.3.015 Hyperhomocysteinemia, methylenetetrahydrofolate reductase 677C→T polymorphism and psychiatric disorders in children

The novel 1,4-dihydropyridine derivatives containing the cationic pyridine moiety at the position 4, and the N-propargyl group as a substituent at position 1 of the 1,4-DHP cycle were designed, synthesised, and assessed in biological tests. Among all the novel compounds, the 4-(N-dodecyl) pyridinium group-containing compounds 11 (without the N-propargyl group) and 12 (with the N-propargyl group) demonstrated the highest calcium antagonistic properties against neuroblastoma SH-SY5Y (IC50 about 5–14 μM) and the vascular smooth muscle A7r5 cell (IC50 – 0.6–0.7 μM) lines, indicating that they predominantly target the L-type calcium channels. These compounds showed a slight total antioxidant activity. At concentrations close to those of L-type calcium channel blocking ones, compound 12 did not affect mitochondrial functioning; also, no toxicity was obtained in vivo. The N-propargyl group as a substituent at position 1 of the 1,4-DHP cycle did not essentially influence the compounds’ activity. The 4-(N-dodecyl) pyridinium moiety-containing compounds can be considered as prototype molecules for further chemical modifications and studies as cardioprotective/neuroprotective agents.

DHEA attenuates PDGF-induced phenotypic proliferation of vascular smooth muscle A7r5 cells through redox regulation

It is known that dehydroepiandrosterone (DHEA) inhibits a phenotypic switch in vascular smooth muscle cells (VSMC) induced by platelet-derived growth factor (PDGF)-BB. However, the mechanism behind the effect of DHEA on VSMC is not clear. Previously we reported that low molecular weight-protein tyrosine phosphatase (LMW-PTP) dephosphorylates PDGF receptor (PDGFR)-β via a redox-dependent mechanism involving glutathione (GSH)/glutaredoxin (GRX)1. Here we demonstrate that the redox regulation of PDGFR-β is involved in the effect of DHEA on VSMC. DHEA suppressed the PDGF-BB-dependent phosphorylation of PDGFR-β. As expected, DHEA increased the levels of GSH and GRX1, and the GSH/GRX1 system maintained the redox state of LMW-PTP. Down-regulation of the expression of LMW-PTP using siRNA restored the suppression of PDGFR-β-phosphorylation by DHEA. A promoter analysis of GRX1 and γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme of GSH synthesis, showed that DHEA up-regulated the transcriptional activity at the peroxisome proliferator-activated receptor (PPAR) response element, suggesting PPARα plays a role in the induction of GRX1 and γ-GCS expression by DHEA. In conclusion, the redox regulation of PDGFR-β is involved in the suppressive effect of DHEA on VSMC proliferation through the up-regulation of GSH/GRX system.

Polyamine synthesis inhibition induces S phase cell cycle arrest in vascular smooth muscle cells

Polyamines are important for cell growth and proliferation and they are formed from arginine and ornithine via arginase and ornithine decarboxylase (ODC). Arginine may alternatively be metabolised to NO via NO synthase. Here we study if vascular smooth muscle cell proliferation can be reversed by polyamine synthesis inhibitors and investigate their mechanism of action. Cell proliferation was assessed in cultured vascular smooth muscle A7r5 cells and in endothelium-denuded rat arterial rings by measuring [3H]-thymidine incorporation and by cell counting. Cell cycle phase distribution was determined by flow cytometry and polyamines by HPLC. Protein expression was determined by Western blotting. The ODC inhibitor DFMO (1-10 mM) reduced polyamine concentration and attenuated proliferation in A7r5 cells and rat tail artery. DFMO accumulated cells in S phase of the cell cycle and reduced cyclin A expression. DFMO had no effect on cell viability and apoptosis as assessed by fluorescence microscopy. Polyamine concentration and cellular proliferation were not affected by the arginase inhibitor NOHA (100-200 microM) and the NO synthase inhibitor L-NAME (100 microM). Lack of effect of NOHA was reflected by absence of arginase expression. Polyamine synthesis inhibition attenuates vascular smooth muscle cell proliferation by reducing DNA synthesis and accumulation of cells in S phase, and may be a useful approach to prevent vascular smooth muscle cell proliferation in cardiovascular diseases.

Vascular metabolic dysfunction and lipotoxicity

The purpose of this study was to determine the role of lipotoxicity in vascular smooth muscle (VSM). C(1)-BODIPY 500/510 C(12) used to assess the ability of VSM A7r5 cells to transport long-chain fatty acids showed that lipid transport did not appear to limit metabolism. Thin layer chromatography revealed that storage of transported fatty acid occurred primarily as mono- and diglycerides and fatty acids but not as triglycerides. We used lipid-induced apoptosis as a measure of lipotoxicity and found that 1.5 mM palmitate (6.8:1) bound to albumin resulted in a 15-fold increase in the number of apoptotic cells compared to the control at 24 hours. This apoptosis did not seem to be due to an increase in reactive oxygen species (ROS) since VSM cells incubated in palmitate showed less ROS production than cells incubated in albumin only. Similar exposure to oleate did not significantly increase the number of apoptotic cells compared to the control. Oleate actually significantly attenuated the apoptosis induced by palmitate, suggesting that unsaturated fatty acids have a protective effect on cells undergoing palmitate-induced apoptosis. These results suggest that vascular smooth muscle is vulnerable to lipotoxicity and that this lipotoxicity may play a role in the development of atherosclerosis.

Vascular Metabolic Dysfunction and Lipotoxicity

The purpose of this study was to determine the role of lipotoxicity in vascular smooth muscle (VSM). C(1)-BODIPY 500/510 C(12) used to assess the ability of VSM A7r5 cells to transport long-chain fatty acids showed that lipid transport did not appear to limit metabolism. Thin layer chromatography revealed that storage of transported fatty acid occurred primarily as mono- and diglycerides and fatty acids but not as triglycerides. We used lipid-induced apoptosis as a measure of lipotoxicity and found that 1.5 mM palmitate (6.8:1) bound to albumin resulted in a 15-fold increase in the number of apoptotic cells compared to the control at 24 hours. This apoptosis did not seem to be due to an increase in reactive oxygen species (ROS) since VSM cells incubated in palmitate showed less ROS production than cells incubated in albumin only. Similar exposure to oleate did not significantly increase the number of apoptotic cells compared to the control. Oleate actually significantly attenuated the apoptosis induced by palmitate, suggesting that unsaturated fatty acids have a protective effect on cells undergoing palmitate-induced apoptosis. These results suggest that vascular smooth muscle is vulnerable to lipotoxicity and that this lipotoxicity may play a role in the development of atherosclerosis.